Hippuricase gene

ABSTRACT

A nucleic acid molecule having a sequence encoding benzoyl-glycine aminohydrolase, commonly known as hippuricase, of Camplylobacter jejuni is provided. Methods are disclosed for detecting C. jejuni in a biological sample by determining the presence of hippuricase or a nucleic acid molecule encoding hippuricase in the sample.

This is a Divisional of application Ser. No. 08/485,216, filed Jun. 7,1995 now U.S. Pat. No. 5,695,960, which is a Continuation ofPCT/CA94/00270, filed May 13, 1994, now International Publication No. WO94/26907, published Nov. 24, 1994, which is a Continuation-In-Part ofapplication Ser. No. 08/061,696, filed May 14, 1993 (abandoned), whichapplication(s) are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a nucleic acid molecule having asequence encoding benzoyl-glycine aminohydrolase, commonly known ashippuricase, of Campylobacter jejuni, or an oligonucleotide fragmentthereof. The nucleic acid molecule of the invention can be used toproduce polypeptides having part or all of the primary structuralconformation and the enzymatic activity of hippuricase.

The nucleic acid molecule of the invention also permits selection of DNAand amino acid sequences unique to polypeptides having the primarystructural confirmation and the enzymatic activity of hippuricase. NovelDNA segments or proteins can thus be constructed which contain theunique DNA and amino acid sequences. The invention also relates to usesof the nucleic acid molecules and the polypeptides of the invention.

BACKGROUND OF THE INVENTION

Campylobacter jejuni (C. jejuni), a gram-negative microaerophilicbacterium, is a leading cause of bacterial diarrhea and enterocolitis inchildren and adults in both developing and developed countries (Walker RI et al, Microbiol. Rev. 50 (1): 81-94, 1986; Kim N W et al, J.Bacteriol. 174 (11):3494-3498, 1992; Chan V L and Bingham H L, Gene101:51-58, 1991). Clinical symptoms of Campylobacter infections rangefrom watery diarrhea to inflammatory dysentery and bloody diarrhea(Cover TL and Blaser N J, Ann. Rev. Ned. 40:269-285, 1989; Walker R I etal, supra). Complications from C. jejuni infections have includedGuillain-Barre syndrome, a neurological disease which may lead torespiratory paralysis and death, toxic megacolon, acute mesentericadenitis syndrome, and reactive arthritis (Kaldor J and Speed B R,British Medical J. 288:1867-1870, 1984; Johnson K et al, Acta. Med.Scand. 214:165-168, 1983; Walker R I et al, supra).

Campylobacter jejuni is commonly found in surface water, in animals suchas cattle, sheep, goats, swine and poultry, in industrial wastes, and inmany different types of foods including unpasteurized dairy products.Human pets such as dogs, cats and birds may also be infected with C.jejuni and may transmit the bacterium to humans. (Cover T L and Blaser MJ, Ann. Rev. Med. 40:269-285, 1989; and Penner, J. L., Clin. Micro. Rev.1:157-172, 1988).

A number of different strategies have been developed for detecting andidentifying C. jejuni in food samples, water, and environmental andclinical specimens. C. jejuni has been differentiated from otherpathogenic camplylobacteria such as C. coli and C. lari, by its abilityto hydrolyze hippurate (Cowan S T, Int. Bull. Bact. Nom. Tax. 5:97,1955). The enzyme benzoyl-glycine aminohydrolase, commonly known ashippuricase, is responsible for cleaving N-benzoylglycine (hippurate)into benzoic acid and glycine. The enzyme is present in C. jejuni and itis either absent or non-functional in other campylobacteria, includingC. coli and C. lari.

Hippuricase activity has been shown in a variety of microorganismsincluding Actinobacillus, Aerobacter aerogenes, Aerococcus viridans,Campylobacter jejuni, certain Enterobacteriaceae, some species in thegenera Bacillus, Benekea, Corynebacterium, Listeria, Pediococcus,Pseudomonas, Streptococcus, Mycobacterium, and Nocardia, and in thefungi Fusarium semiticum, and Streptomyces. Hippuricase fromStreptococcus (Braunstein H et al., Am. J. Clin. Pathol. 51:207, 1969;Facklam R R et al., Appl. Microbiol. 27:107, 1974), Pseudomonas (Kamedaet al., Chem. Pharm. Bull. Tokyo 16:1023, 1968), and Fusarium semicticum(Rohr M., Monatshefte fur Chemie 99:2255-2277, 1968), has been partiallycharacterized. The molecular weight has not been determined but it isestimated to be between 70,000 to 100,000. The protein has been shown tobe antigenic and antisera has been prepared to the streptococcal enzyme.Only fragmentary evidence on hippuricase is available for otherorganisms. It has been postulated that hippuricase is used bymicroorganisms as a mechanism of generating either an amino acid orbenzoic acid as a substrate for metabolism.

Methods currently used to detect hippuricase activity in Campylobacterjejuni include the standard procedure for streptococci by Hwang andEderer (Hwang M and Ederer G M, J. Clin. Microbiol. 1:114, 1975; EdbergS C and Samuels S, J. Clin. Microbiol. 3:49, 1976) based on thedetection of glycine using a ninhydrin reagent. Hippuricase activity hasalso been detected through the determination of the second product,benzoic acid (Ottow J C G, J. Appl. Bacteriol. 37:15, 1974; Ayers andRupp, supra, 1922).

These methods are not adequate to allow accurate detection of C. jejuni.For example, C. coli may yield weak hippuricase reactions, and C. jejunimay give weak or no hippuricase activity (Totten P A et al., J. Clin.Microbiol. 25:1747-1752, 1987). The currently used methods for detectinghippuricase activity are also cumbersome due to the need to culture theorganisms prior to testing. Further, nonconventional means such asgas-liquid chromatography must be used to quantitate the hydrolysedbenzoic acid product in very weak reactions (Bar W and Fricke G, J.Clin. Microbiol. 25:1776-1778, 1987; Wallace P L et al, J. Clin.Microbiol. 25:3766-1768, 1987).

DNA probes have been developed for detection and identification of C.jejuni and other campylobacteria. None of the probes have been fullycharacterized and the nature of the gene products and their functionsare unknown. Romaniuk and Trust (Mol. Cell. Probes 3:133-142, 1989) usedpartial rRNA sequence information of campylobacteria to developoligonucleotide probes to 16 S ribosomal RNA. One of these three oligoprobes is reported to specifically identify C. jejuni and C. lari, andthe other two are reported to be specific for C. jejuni, C. coli, and C.lari. Barns et al (European Patent Application No. 89306594.6, publishedon Jan. 10, 1990 as No. 0,350,205) disclose small nucleic acid probeswhich are reported to be capable of specifically hybridizing toribosomal RNA of C. jejuni, C. coli and C. laridis and not to rRNA orrRNA genes of Pseudomonas aeroginosa, E. coli, or Salmonellatyphimurium.

Taylor and Hiratsuka (Mol. Cell. Probes 4:261-271, 1989) developed twoDNA probes using cloned C. jejuni genomic fragments obtained byscreening a lambda gtll library with an antiserum prepared against a 46kD major outer membrane protein of C. jejuni One of the probes (pDT1728)is reported to be C. jejuni specific, while the other (pDT1719) isreported to detect C. jejuni and C. coli but has lower sensitivity forthe latter. Rashtchian (U.S. Pat. No. 4,785,086) describes a DNA probecapable of hybridizing to DNA of at least 80% of C. jejuni bacteria, aswell as DNA in other campylobacteria.

Blaser et al. (U.S. Pat. No. 5,200,344) disclose antigenic compositions,and antibodies against the antigenic compositions, for use in diagnostictesting for C. jejuni or C. coli. The antigens in the compositions areobtained by acid extraction of surface antigens of C. jejuni and/or C.coli. The antigenic compositions described by Blaser et al. are notcapable of differentiating C. jejuni and C. coli.

Antibody probes developed for campylobacteria are limited to theserotype antisera used to establish the epidemiological relationshipbetween various Campylobacter isolates. These antibody probes are usedonly after the isolates have been identified as belonging to the genusby using the conventional methods of biochemical reactions, morphology,cultural and Gram reactions. The serotyping systems of Penner and Liorutilize antibody reactions against heat stable and heat-labile antigensrespectively (Penner J L, Clin. Micro. Rev. 1:157-172, 1988; and Lior Het al., J. Clin. Microbiol. 15:761-768, 1982). The thermostable antigenshave been proven to be the lipopolysaccharide antigens located in theouter membrane of the organism, and are detected through passivehemagglutination of erythrocytes. The Lior system uses antisera thathave been absorbed with thermostable antigens of the homologousserostrain, and detects thermolabile antigens by means of slideagglutination of cell suspensions. The mixture of heat-labile antigensis as yet uncharacterized.

SUMMARY OF THE INVENTION

The present inventors have isolated and identified a nucleotide sequencefrom C. jejuni encoding a protein having hippuricase activity. They havealso determined that the sequence is specific for Campylobacter jejuniand it is not detectable by DNA hybridization in any other species ofCampylobacter. Therefore, the present invention permits the preparationof probes and specific antibodies which will be useful in diagnostictesting to specifically differentiate C. jejuni from othercampylobacteria. The use of a probe based on the identified hippuricasenucleotide sequence of the invention, or antibodies prepared using ahippuricase protein of the invention, will provide highly specific,sensitive, and rapid means to detect the presence of C. jejuni insamples.

The high incidence of C. jejuni infections in children and adults indeveloping and developed countries and the significant costs associatedwith the treatment of the infections and complications arisingtherefrom, make it highly desirable to have a rapid and specific test todetect and monitor C. jejuni in clinical specimens and in potentialsources of the organism. The present invention is important in itsability to specifically detect C. jejuni and in not requiringpropagation of the organisms.

Accordingly, the present invention provides a purified and isolatednucleic acid molecule comprising a sequence encoding hippuricase ofCampylobacter jejuni, or an oligonucleotide fragment of the sequencewhich is unique to hippuricase of C. jejuni.

In a preferred embodiment, a purified and isolated nucleic acid moleculeis provided having a sequence which encodes hippuricase having an aminoacid sequence as shown in FIG. 1 and in the Sequence Listing as SEQ. ID.NO. 1 and No. 2. Most preferably, the purified and isolated nucleic acidmolecule comprises (a) a nucleic acid sequence as shown in SEQ ID NO:1and FIG. 1, wherein T can also be U; (b) nucleic acid sequencescomplementary to (a); (c) nucleic acid sequences which are at least 85%homologous to (a); or, (d) a fragment of (a) or (b) that is at least 15bases, preferably 20 to 30 bases, and which will hybridize to (a) or (b)under stringent hybridization conditions.

The nucleic acid molecules of the invention may be inserted into anappropriate expression vector, i.e. a vector which contains thenecessary elements for the transcription and translation of the insertedprotein-coding sequence. Accordingly, recombinant DNA molecules adaptedfor transformation of a host cell may be constructed which comprise anucleic acid molecule of the invention operatively linked to anexpression control sequence. A transformant host cell including arecombinant molecule of the invention is also provided. Still further,this invention provides plasmids which comprise the recombinantmolecules of the invention.

The present invention further relates to an avirulent strain ofCampylobacter jejuni comprising an avirulent bacterial carrier straintransformed with a recombinant molecule of the invention, and a vaccinecomposition comprising a bacterial carrier strain transformed with arecombinant molecule of the invention.

The invention also provides a method of preparing hippuricase utilizinga nucleic acid molecule of the invention. The method comprises culturinga transformant host cell including a recombinant molecule comprising aDNA segment of the invention and an expression control sequenceoperatively linked to the DNA segment, in a suitable medium untilhippuricase is formed and thereafter isolating the hippuricase.

The invention still further provides a purified and isolated polypeptidehaving part or all of the primary structural confirmation (ie.continuous sequence of amino acid residues) and the enzymatic activityof hippuricase. In a preferred embodiment the polypeptide has an aminoacid sequence as shown in FIG. 1 and in the Sequence Listing as SEQ IDNO:1 and NO:2, or a sequence having between 97 and 100 percent homologythereto.

The invention also relates to an antibody specific for an epitope of apolypeptide of the invention, preferably a monoclonal antibody andmethods for preparing the antibodies. A method for detecting C. jejuniin a sample is provided comprising assaying for hippuricase in thesample. In an embodiment of the invention the method comprisescontacting the sample with an antibody of the invention which is capableof being detected after it becomes bound to hippuricase in the sample,and measuring the amount of antibody bound to hippuricase in the sample,or unreacted antibody.

A kit for detecting Campylobacter jejuni in a sample comprising anantibody of the invention, preferably a monoclonal antibody anddirections for its use is also provided. The kit may also containreagents which are required for binding of the antibody to hippuricasein the sample.

The nucleic acid molecules of the invention allow those skilled in theart to construct nucleotide probes for use in the detection ofnucleotide sequences in samples such as biological, food, orenvironmental samples. The nucleotide probes may be used to detectnucleotide sequences that encode polypeptides related to or analogous tothe hippuricase polypeptides of the invention.

Accordingly, the invention provides a method for detecting the presenceof a nucleic acid molecule having a sequence encoding a polypeptiderelated to or analogous to a polypeptide of the invention, comprisingcontacting the sample with a nucleotide probe which hybridizes with thenucleic acid molecule, to form a hybridization product under conditionswhich permit the formation of the hybridization product, and assayingfor the hybridization product.

The invention further provides a kit for detecting the presence of anucleic acid molecule having a sequence encoding a polypeptide relatedto or analogous to a polypeptide of the invention, comprising anucleotide probe which hybridizes with the nucleic acid molecule,reagents required for hybridization of the nucleotide probe with thenucleic acid molecule, and directions for its use.

The nucleic acid molecules of the invention also permit theidentification and isolation, or synthesis, of nucleotide sequenceswhich may be used as primers to amplify a nucleic acid molecule of theinvention, for example in the polymerase chain reaction (PCR).

Accordingly, the invention relates to a method of determining thepresence of a nucleic acid molecule having a sequence encodinghippuricase or a predetermined part of hippuricase in a sample,comprising treating the sample with primers which are capable ofamplifying the nucleic acid molecule, in a polymerase chain reaction toform amplified sequences, under conditions which permit the formation ofamplified sequences, and, assaying for amplified sequences.

The invention further relates to a kit for determining the presence of anucleic acid molecule having a sequence encoding hippuricase or apredetermined part of hippuricase in a sample, comprising primers whichare capable of amplifying the nucleic acid molecule in a polymerasechain reaction to form amplified sequences, reagents required foramplifying the nucleic acid molecule thereof in an amplificationreaction, preferably the polymerase chain reaction, means for assayingthe amplified sequences, and directions for its use.

The nucleic acid molecules of the invention may also be used to assayfor a substance which specifically affects Campylobacter jejuni.Accordingly, the invention provides a method for assaying for asubstance that affects hippuricase activity comprising providing apolypeptide of the invention, incubating with a substrate of thepolypeptide, and a test substance which is suspected of affectinghippuricase, and determining the effect of the substance by comparing toa control. The method may be used, for example, to assay for a substancewhich affects the growth or pathogenicity of Campylobacter jejuni.

BRIEF DESCRIPTION OF DRAWINGS

The invention will now be described in relation to the drawings:

FIG. 1 is the nucleotide sequence of the hippuricase gene ofCampylobacter jejuni;

FIG. 2 is a restriction map of recombinant plasmid pHIPPO containing thehippuricase gene of C. Jejuni;

FIG. 3 shows the location of the hippuricase ene on the physical map ofC. jejuni strain TGH9011;

FIG. 4 shows the results of the maxicell analysis of the protein productof the hippuricase gene of he invention;

FIG. 5 is a Southern hybridization of chromosomal DNA of hippuricasenegative C. jejuni isolates; and

FIG. 6 is a Southern hybridization of chromosomal DNA of C. coli and C.jejuni isolates.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As hereinbefore mentioned, the present inventors have identified andsequenced a DNA sequence of C. jejuni encoding hippuricase. The DNAsequence and deduced amino acid sequence are shown in the SequenceListing as SEQ ID NO:1 and NO:2.

Nucleic acid molecules of the present invention encoding hippuricase orrelated or analogous polypeptides may be isolated and sequenced, byselectively amplifying the region of a hippuricase gene, or related oranalogous genes, using the polymerase chain reaction method and genomicDNA. It is possible to design synthetic oligonucleotide primers from thesequence shown in the Sequence Listing as SEQ ID NO:1 for use in PCR andfor screening genomic libraries. An amplified fragment can be cloned andcharacterized by DNA sequence analysis. Nucleic acid molecules of thepresent invention encoding hippuricase may also be constructed bychemical synthesis and enzymatic ligation reactions using proceduresknown in the art.

It will be appreciated that the invention includes nucleotide or aminoacid sequences which have substantial sequence homology with thenucleotide and amino acid sequences shown in the Sequence Listing as SEQID NO:1 and NO:2. The term "sequences having substantial sequencehomology" means those nucleotide and amino acid sequences which haveslight or inconsequential sequence variations from the sequencesdisclosed in the Sequence Listing as SEQ ID NO:1 and NO:2 i.e. thehomologous sequences function in substantially the same manner toproduce substantially the same polypeptides as the actual sequences. Thevariations may be attributable to local mutations or structuralmodifications. It is expected that a sequence having 85-90% sequencehomology with the DNA sequence of the invention will provide afunctional hippuricase polypeptide.

Nucleic acid sequences having substantial sequence homology includenucleic acid sequences having at least 85%, preferably at least 90%homology with the nucleic acid sequence as shown in SEQ. ID. NO.:1 andin FIG. 1; and fragments thereof having at least 15 to 30, preferably atleast 15 bases, most preferably 20 to 30, which will hybridize to thesesequences under stringent hybridization conditions. Stringenthybridization conditions are those which are stringent enough to providespecificity, reduce the number of mismatches and yet are sufficientlyflexible to allow formation of stable hybrids at an acceptable rate.Such conditions are known to those skilled in the art and are described,for example, in Sambrook, et al, (1989, Molecular Cloning, A LaboratoryManual, Cold Spring Harbor). By way of example only, stringenthybridization with short nucleotides may be carried out at 5-100 belowthe T_(m) using high concentrations of probe such as 0.01-1.0 pmole/ml.

The invention further provides amino acid sequences which havesubstantial homology with the amino acid sequence shown in SEQ ID NO:2and in FIG. 1. Substantially homologous sequences include sequenceshaving at least 97% sequence homology.

It will also be appreciated that a double stranded nucleotide sequencecomprising a nucleic acid molecule of the invention, hydrogen bonded toa complementary nucleotide base sequence, an RNA made by transcriptionof this doubled stranded nucleotide sequence, and an antisense strand ofa nucleic acid molecule of the invention or an oligonucleotide fragmentof the nucleic acid molecule, are contemplated within the scope of theinvention.

Analysis of the complete nucleotide and amino acid sequences of theprotein of the invention using the procedures of Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual. Cold Spring Harbour LaboratoryPress may allow determination of the initiation codon and untranslatedsequences of the hippuricase gene. The transcription regulatorysequences of the gene may be determined by analyzing fragments of theDNA for their ability to express a reporter gene such as the bacterialgene lacZ. Primer extension using reverse transcriptase may also be usedto determine the initiation site.

In an embodiment of the invention, an antisense sequence of a nucleicacid molecule of the invention is provided. An antisense sequence isconstructed by inverting the sequence of a nucleic acid molecule of theinvention, relative to its normal presentation for transcription.Preferably, an antisense sequence is constructed by inverting a regionpreceding the initiation codon or an unconserved region. The antisensesequences may be constructed using chemical synthesis and enzymaticligation reactions using procedures known in the art.

A number of unique restriction sequences for restriction enzymes areincorporated in the nucleic acid molecule identified in the SequenceListing as SEQ ID NO:1, and these provide access to nucleotide sequenceswhich code for polypeptides unique to the hippuricase polypeptide of theinvention. Nucleotide sequences unique to hippuricase of C. jejuni orisoforms thereof, can also be constructed by chemical synthesis andenzymatic ligation reactions carried out by procedures known in the art.

The nucleic acid molecules of the invention, allow those skilled in theart to construct nucleotide probes for use in the detection ofnucleotide sequences in biological materials, such as feces, blood orother bodily fluids or tissues from humans or animals such as mammalsand poultry, in foods such as dairy products most particularly milk andpoultry, and in environmental samples such as water and industrialwastes.

A nucleotide probe may be labelled with a detectable marker such as aradioactive label which provides for an adequate signal and hassufficient halflife such as 32p, 3 H, 14 C or the like. Other detectablemarkers which may be used include antigens that are recognized by aspecific labelled antibody, fluorescent compounds, enzymes, antibodiesspecific for a labelled antigen, and chemiluminescent compounds. Anappropriate label may be selected having regard to the rate ofhybridization and binding of the probe to the nucleotide to be detectedand the amount of nucleotide available for hybridization.

The nucleotide probes may be used to detect genes that encodepolypeptides related to or analogous to hippuricase of the invention.

Accordingly, the present invention also relates to a method of detectingthe presence of nucleic acid molecules encoding a polypeptide related toor analogous to hippuricase of the invention in a sample comprisingcontacting the sample under hybridization conditions with one or more ofthe nucleotide probes of the invention labelled with a detectablemarker, and determining the degree of hybridization between the nucleicacid molecule in the sample and the nucleotide probes.

Hybridization conditions which may be used in the method of theinvention are known in the art and are described for example in SambrookJ, Fritch E F, Maniatis T. In: Molecular Cloning, A Laboratory Manual,1989. (Nolan C, Ed.), Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. The hybridization product may be assayed using techniquesknown in the art. The nucleotide probe may be labelled with a detectablemarker as described herein and the hybridization product may be assayedby detecting the detectable marker or the detectable change produced bythe detectable marker.

The nucleic acid molecule of the invention also permits theidentification and isolation, or synthesis of nucleotide sequences whichmay be used as primers to amplify a nucleic acid molecule of theinvention, for example in the polymerase chain reaction (PCR) which isdiscussed in more detail below. The primers may be used to amplify thegenomic DNA of other bacterial species known to possess hippuricaseactivity such as Bacillus subtilis and Streptococcus faecalis. The PCRamplified sequences can be examined to determine the relationshipbetween the various hippuricase genes.

The length and bases of the primers for use in the PCR are selected sothat they will hybridize to different strands of the desired sequenceand at relative positions along the sequence such that an extensionproduct synthesized from one primer when it is separated from itstemplate can serve as a template for extension of the other primer intoa nucleic acid of defined length.

Primers which may be used in the invention are oligonucleotides i.e.molecules containing two or more deoxyribonucleotides of the nucleicacid molecule of the invention which occur naturally as in a purifiedrestriction endonuclease digest or are produced synthetically usingtechniques known in the art such as for example phosphotriester andphosphodiester methods (See Good et al Nucl. Acid Res 4:2157, 1977) orautomated techniques (See for example, Conolly, B. A. Nucleic Acids Res.15:15 (7): 3131, 1987). The primers are capable of acting as a point ofinitiation of synthesis when placed under conditions which permit thesynthesis of a primer extension product which is complementary to theDNA sequence of the invention i.e. in the presence of nucleotidesubstrates, an agent for polymerization such as DNA polymerase and atsuitable temperature and pH. Preferably, the primers are sequences thatdo not form secondary structures by base pairing with other copies ofthe primer or sequences that form a hair pin configuration. The primermay be single or double-stranded. When the primer is double-stranded itmay be treated to separate its strands before using to prepareamplification products. The primer preferably contains between about 7and 25 nucleotides.

The primers may be labelled with detectable markers which allow fordetection of the amplified products. Suitable detectable markers areradioactive markers such as P-32, S-35, I-125, and H-3, luminescentmarkers such as chemiluminescent markers, preferably luminol, andfluorescent markers, preferably dansyl chloride,fluorcein-5-isothiocyanate, and 4-fluor-7-nitrobenz-2-axa-1,3 diazole,enzyme markers such as horseradish peroxidase, alkaline phosphatase,β-galactosidase, acetylcholinesterase, or biotin.

It will be appreciated that the primers may contain non-complementarysequences provided that a sufficient amount of the primer contains asequence which is complementary to a nucleic acid molecule of theinvention or oligonucleotide sequence thereof, which is to be amplified.Restriction site linkers may also be incorporated into the primersallowing for digestion of the amplified products with the appropriaterestriction enzymes facilitating cloning and sequencing of the amplifiedproduct.

In an embodiment of the invention a method of determining the presenceof a nucleic acid molecule having a sequence encoding hippuricase or apredetermined oligonucleotide fragment thereof in a sample, is providedcomprising treating the sample with primers which are capable ofamplifying the nucleic acid molecule or the predeterminedoligonucleotide fragment thereof in a polymerase chain reaction to formamplified sequences, under conditions which permit the formation ofamplified sequences and, assaying for amplified sequences.

The polymerase chain reaction refers to a process for amplifying atarget nucleic acid sequence as generally described in Innis et al,Academic Press, 1990 in Mullis el al., U.S. Pat. No. 4,863,195 andMullis, U.S. Pat. No. 4,683,202 which are incorporated herein byreference. Conditions for amplifying a nucleic acid template aredescribed in M. A. Innis and D. H. Gelfand, PCR Protocols, A Guide toMethods and Applications M. A. Innis, D. H. Gelfand, J. J. Sninsky andT. J. White eds, pp3-12, Academic Press 1989, which is aleo incorporatedherein by reference.

The process described by Mullis amplifies any desired specificnucleotide sequence contained in a nucleic acid or mixture thereof. Theprocess involves treating separate complementary strands of thenucleotide sequence to be amplified with two oligonucleotide primerswhich are extended under suitable conditions to form complementaryprimer extension products which act as templates for synthesizing thenucleotide sequence. The primers are selected so that they aresufficiently complementary to different strands of each specificnucleotide sequence to be amplified. The steps of the PCR reaction maybe carried out sequentially or simultaneously and the steps may berepeated until the desired level of amplification is obtained.

The amplified products can be isolated and distinguished based on theirrespective sizes using techniques known in the art. For example, afteramplification, the DNA sample can be separated on an agarose gel andvisualized, after staining with ethidium bromide, under ultra violet (W)light. DNA may be amplified to a desired level and a further extensionreaction may be performed to incorporate nucleotide derivatives havingdetectable markers such as radioactive labelled or biotin labellednucleoside triphosphates. The primers may also be labelled withdetectable markers. The detectable markers may be analyzed byrestriction and electrophoretic separation or other techniques known inthe art.

The conditions which may be employed in the methods of the inventionusing PCR are those which permit hybridization and amplificationreactions to proceed in the presence of DNA in a sample and appropriatecomplementary hybridization primers. Conditions suitable for thepolymerase chain reaction are generally known in the art. For example,see M. A. Innis and D. H. Gelfand, PCR Protocols, A guide to Methods andApplications M. A. Innis, D. H. Gelfand, J. J. Sninsky and T. J. Whiteeds, pp3-12, Academic Press 1989, which is incorporated herein byreference. Preferably, the PCR utilizes polymerase obtained from thethermophilic bacterium Thermus aquatics (Taq polymerase, GeneAmp Kit,Perkin Elmer Cetus) or other thermostable polymerase may be used toamplify DNA template strands.

It will be appreciated that other techniques such as the Ligase ChainReaction (LCR) and NASBA may be used to amplify a nucleic acid moleculeof the invention. In LCR, two primers which hybridize adjacent to eachother on the target strand are ligated in the presence of the targetstrand to produce a complementary strand (Barney in "PCR Methods andApplications", August 1991, Vol.1 (1), page 5, and European PublishedApplication No. 0320308, published Jun. 14, 1989). NASBA is a continuousamplification method using two primers, one incorporating a promotersequence recognized by an RNA polymerase and the second derived from thecomplementary sequence of the target sequence to the first primer (U.S.Ser. No. 5,130,238 to Malek).

A nucleic acid molecule of the present invention may be incorporated ina known manner into a recombinant molecule which ensures good expressionof the hippuricase polypeptide or part thereof. In general, arecombinant molecule of the invention contains a nucleic acid moleculeof the invention and a suitable transcriptional or translationalregulatory element operatively linked to the nucleic acid molecule.Suitable regulatory elements may be derived from a variety of sources,including bacterial, fungal, viral, mammalian or insect genes. Selectionof appropriate regulatory elements is dependent on the host cell chosen,and may be readily accomplished by one of ordinary skill in the art.Examples of regulatory elements include: a transcriptional promoter andenhancer or RNA polymerase binding sequence, a ribosomal bindingsequence, including a translation initiation signal. Additionally,depending on the host cell chosen and the vector employed, other geneticelements, such as an origin of replication, additional DNA restrictionsites, enhancers, sequences conferring inducibility of transcription,and selectable markers, may be incorporated into the expression vector.Preferably the regulatory sequences of the hippuricase gene are used asthe regulatory elements in a recombinant molecule of the invention. Theregulatory sequences of the hippuricase gene may be isolated asdescribed above. A nucleic acid molecule of the invention, may beincorporated into a plasmid vector. The plasmid vectors may be designedto facilitate subsequent purification of the encoded protein or partsthereof by affinity chromatography. For example, gene fusion systemssuch as the glutathione S-transferase gene fusion system may beutilized.

The hippuricase polypeptide or isoforms or parts thereof, may beobtained by expression in a suitable host cell using techniques known inthe art. Suitable host cells include prokaryotic or eukaryotic organismsor cell lines, for example bacterial, mammalian, yeast, or other fungi,viral, plant or insect cells. Methods for transforming or transfectingcells to express foreign DNA are well known in the art (See for example,Itakura et al., U.S. Pat. No. 4,704,362; Hinnen et al., PNAS USA75:1929-1933, 1978; Murray et al., U.S. Pat. No. 4,801,542; Upshall etal., U.S. Pat. No. 4,935,349; Hagen et al., U.S. Pat. No. 4,784,950;Axel et al., U.S. Pat. No. 4,399,216; Goeddal et al., U.S. Pat. No.4,766,075; and Sambrook et al Molecular Cloning: A Laboratory Manual 2ndEd, Cold Spring Harbor Laboratory Press, 1989, all of which areincorporated herein by reference).

Bacterial hosts suitable for carrying out the present invention includeE. coli as well as many other bacterial species well known to one ofordinary skill in the art. Representative examples of bacterial hostcells include E. coli, Streptococcus, Campylobacter, and Bacillussubtilus.

Bacterial expression vectors preferably comprise a promoter whichfunctions in the host cell, one or more selectable phenotypic markers,and a bacterial origin of replication. Representative promoters includethe β-lactamase (pencillinase) and lactose promoter system (see Chang etal., Nature 275:615, 1978), the trp promoter (Nichols and Yanofsky,Meth. in Enzymology 101:155, 1983) and the tac promoter (Russel el al.,Gene 20: 231, 1982). Examples of selectable markers are variousantibiotic resistance markers such as the kanamycin or ampicillinresistance genes. Nany plasmids suitable for transforming host cells arewell known in the art, including pBR322 (see Bolivar et al., Gene 2:95,1977), the pUC plasmids pUC18, pUC19, pUC118, pUC119 (see Messing, Neth.in Enzymology 101:20-77, 1983 and Vieira and Messing, Gene 19:259-268,1982), and pNH8A, pNH16a, pNH18a and pBluescript M13 (Stratagene, LaJolla, Calif.).

Yeast and fungi host cells suitable for carrying out the presentinvention include, among others Saccharomyces cerevisiae, the generaPinchi or Kluyveromyces and various species of the genus Aspergillus.Suitable expression vectors for yeast and fungi include, among others,YCp50 (ATCC No. 37419) for yeast, and amdS cloning vector pV3 (Turnbull,Bio/Technology 7:169, 1989). Protocols for the transformation of yeastare also well known to those of ordinary skill in the art. For example,transformation may be readily accomplished either by preparation ofspheroplasts of yeast with DNA (see Hinnen et al., PNAS USA 75:1929,1978) or by treatment with alkaline salts such as LiCl (see Itoh et al.,J. Bacteriology 153:163, 1983). Transformation of fungi may also becarried out sing polyethylene glycol as described by Cullen et al.(Bio/Technology 5:369, 1987).

The polypeptides of the invention may be prepared by culturing thehost/vector systems described above, in order to express the recombinantpolypeptides. Recombinantly produced hippuricase or parts thereof, maybe further purified using techniques known in the art such ascommercially available protein concentration systems, by salting out theprotein followed by dialysis, by affinity chromatography, or using anionor cation exchange resins.

In a preferred embodiment, a nucleic acid molecule of the invention maybe cloned into a glutathione S-transferase (GST) gene fusion system forexample the pGEX-1 T, pGEX-2T and pGEX-3X of Pharmacia. The fused genemay contain a strong tac promoter, inducible to a high level ofexpression by IPTG, as a regulatory element. Thrombin or factor Xacleavage sites may be present which allow proteolytic cleavage of thedesired polypeptide from the fusion product. The glutathioneS-transferase-hippuricase fusion protein may be easily purified using aglutathione sepharose 4B column, for example from Pharmacia. The 26 kdglutathione S-transferase polypeptide can be cleaved by thrombin (pGEX-1or pGEX-2T) or factor Xa (pGEX-3X) and resolved from the hippuricasepolypeptide using the same affinity column. Additional chromatographicsteps can be included if necessary, for example Sephadex or DEAEcellulose. The two enzymes may be monitored by protein and enzymaticassays and purity may be confirmed using SDS-PAGE.

The hippuricase protein or parts thereof may also be prepared bychemical synthesis using techniques well known in the chemistry ofproteins such as solid phase synthesis (Merrifield, 1964, J. Am. Chem.Assoc. 85:2149-2154) or synthesis in homogenous solution (Houbenweyl,1987, Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I and II,Thieme, Stuttgart).

Within the context of the present invention, hippuricase polypeptide mayinclude various structural forms of the primary protein which retainbiological activity. For example, hippuricase polypeptide may be in theform of acidic or basic salts or in neutral form. In addition,individual amino acid residues may be modified by oxidation orreduction. Furthermore, various substitutions, deletions or additionsmay be made to the amino acid or nucleic acid sequences, the net effectbeing that biological activity of hippuricase is retained. Due to codedegeneracy, for, example, there may be considerable variation innucleotide sequences encoding the same amino acid.

Mutations in nucleotide sequences constructed for expression ofderivatives of hippuricase polypeptide must preserve the reading framephase of the coding sequences. Furthermore, the mutations willpreferably not create complementary regions that could hybridize toproduce secondary mRNA structures, such as loops or hairpins, whichcould adversely affect translation of the receptor mRNA.

Mutations may be introduced at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes a derivativehaving the desired amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site specific mutagenesisprocedures may be employed to provide an altered gene having particularcodons altered according to the substitution, deletion, or insertionrequired. Deletion or truncation of hippuricase may also be constructedby utilizing convenient restriction endonuclease sites adjacent to thedesired deletion. Subsequent to restriction, overhangs may be filled in,and the DNA relegated. Exemplary methods of making the alterations setforth above are disclosed by Sambrook et al (Molecular Cloning: ALaboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, 1989).

The hippuricase polypeptide of the invention may be expressed in anavirulent bacterial carrier strain such as Salmonella and Shigellastrains. Accordingly, in a further aspect of the invention an avirulentstrain of a C. jejuni is provided comprising an avirulent bacterialcarrier strain transformed with a recombinant molecule of the invention.The avirulent strain may provide the basis for a vaccine compositionwhich may be useful for effecting immunity against diseases caused by C.jejuni. The invention therefore also provides a vaccine compositioncomprising a bacterial carrier strain transformed with a recombinantmolecule of the invention. The vaccine composition may be useful ineffecting immunity against diseases caused by C. jejuni.

The vaccine compositions can be prepared by per se known methods for thepreparation of pharmaceutically acceptable vaccines which can beadministered to patients. The vaccine composition may be in an oral orinjectable form and may include pharmaceutically acceptable vehicles. Onthis basis, the vaccine compositions include, albeit not exclusively,solutions of the bacterial carrier strain transformed with a recombinantmolecule of the invention in association with one or morepharmaceutically acceptable vehicles or diluents, and contained inbuffered solutions with a suitable pH and iso-osmotic with thephysiological fluids. Suitable pharmaceutically acceptable vehicles ordiluents are described, for example, in Remington's PharmaceuticalSciences (Mack Publishing Company, Easton, Pa., U.S.A., 1985).

The hippuricase protein of the invention or parts thereof, may be usedto prepare antibodies. "Antibodies" used herein are understood toinclude polyclonal antibodies, monoclonal antibodies, antibody fragments(e.g., Fab' and F(ab')2 ) and recombinantly produced partners.Antibodies may be prepared which bind a distinct epitope in anunconserved region of the protein; for example the nucleotide bindingfolds.

Conventional methods can be used to prepare the antibodies. Monoclonalantibodies may be readily generated using conventional techniques (seeU.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993 which areincorporated herein by reference; see also "Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses", Plenum Press,Kennett, McKearn, and Bechtol (eds.), Cold Spring Harbor LaboratoryPress, 1988, and Goding, J. W., Monoclonal Antibodies: Principles andPractice, 2nd Ed., Academic Press, London, 1986 which are alsoincorporated herein by reference).

Briefly, in one embodiment a subject animal such as a rat or mouse isinjected with a polypeptide of the invention. The polypeptide may beadmixed with an adjuvant such as Freund's complete or incompleteadjuvant in order to increase the resultant immune response. Between oneand three weeks after the initial immunization the animal may bereimmunized with another booster immunization, and tested for reactivityto hippuricase using assays described above. Once the animal'sreactivity to hippuricase has plateaued, the animal is sacrificed, andorgans which contain large numbers of B cells such as the spleen andlymph nodes are harvested.

Cells which are obtained from immunized animals may be immortalized bytransfection with a virus such as the Epstein bar virus (EBV) (seeGlasky and Reading, Hybridoma 8 (4):377-389, 1989). Alternatively,within a preferred embodiment, the harvested spleen and/or lymph nodecell suspensions are fused with a suitable myeloma cell in order tocreate a "hybridoma" which secretes monoclonal antibody. Suitablemyeloma lines include, for example, NS-1 (ATCC No. TIB 18), and P3X63-Ag8.653 (ATCC No. CRL 1580).

Following the fusion, the cells may be placed in culture platescontaining a suitable medium, such as RPMI 1640, or DMEM (Dulbecco'sModified Eagles Medium) (JRH Biosciences, Lenexa, Kans.), as well asadditional ingredients, such as Fetal Bovine Serum (FBS, i.e., fromHyclone, Logan, Utah, or JRH Biosciences). Additionally, the mediumshould contain a reagent which selectively allows for the growth offused spleen and myeloma cells such as HAT (hypoxanthine, aminopterin,and thymidine) (Sigma Chemical Co., St. Louis, Mo.). After about sevendays, the resulting fused cells or hybridomas may be screened in orderto determine the presence of antibodies which are reactive againsthippuricase. A wide variety of assays may be utilized to determine thepresence of antibodies which are reactive against hippuricase, includingfor example Countercurrent Immuno-Electrophoresis, Radioimmunoassays,Radioimmunoprecipitations, Enzyme-Linked Immunosorbent Assays (ELISA),DOT Blot assays, Inhibition or Competition Assays, and sandwich assays(see U.S. Pat. Nos. 4,376,101 and 4,486,530; see also Antibodies: ALaboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor LaboratoryPress, 1988). Following several clonal dilutions and reassays, ahybridoma producing antibodies reactive against a polypeptide of theinvention may be isolated.

Other techniques may also be utilized to construct monoclonal antibodies(see William D. Huse et al., "Generation of a Large CombinationalLibrary of the Immunoglobulin Repertoire in Phage Lambda," Science246:1275-1281, December 1989; see also L. Sastry et al., "Cloning of theImmunological Repertoire in Escherichia coli for Generation ofMonoclonal Catalytic Antibodies: Construction of a Heavy Chain VariableRegion-Specific cDNA Library," Proc. Natl. Acad. Sci. USA 86:5728-5732,August 1989; see also Michelle Alting-Mees et al., "Monoclonal AntibodyExpression Libraries: A Rapid Alternative to Hybridomas," Strategies inMolecular Biology 3:1-9, January 1990; these references describe acommercial system available from Stratacyte, La Jolla, Calif., whichenables the production of antibodies through recombinant techniques).Briefly, mRNA is isolated from a Bell cell population, and utilized tocreate heavy and light chain immunoglobulin cDNA expression libraries inthe yImmunoZap(H) and yImmunoZap(L) vectors. These vectors may bescreened individually or co-expressed to form Fab fragments orantibodies (see Huse et al., supra; see also Sastry et al., supra).Positive plaques may subsequently be converted to a non-lytic plasmidwhich allows high level expression of monoclonal antibody fragments fromE. coli.

Similarly, binding partners may also be constructed utilizingrecombinant DNA techniques to incorporate the variable regions of a genewhich encodes a specifically binding antibody. Within one embodiment,the genes which encode the variable region from a hybridoma producing amonoclonal antibody of interest are amplified using nucleotide primersfor the variable region. These primers may be synthesized by one ofordinary skill in the art, or may be purchased from commerciallyavailable sources. Stratacyte (La Jolla, Calif.) sells primers for mouseand human variable regions including among others, primers for V_(Ha),V_(Hb), V_(Hd), C^(H1), V^(L) and CL regions. These primers may beutilized to amplify heavy or light chain variable regions, which maythen be inserted into vectors such as ImmunoZAP™ H or ImmunoZAP™ L(Stratacyte), respectively. These vectors may then be introduced into E.coli for expression. Mouse-human chimeric monoclonal antibodies may beconstructed using the DNA sequences of the invention in the methoddescribed by Hirofumi Hamada et al., Cancer Research 50, 3167-3171, 1991and Yasuhiko Nishiola et al., Jpn. J. Cancer Res. 83, 644-649, 1192.

Utilizing these techniques, large amounts of a single-chain proteincontaining a fusion of the VH and VL domains may be produced (see Birdet al., Science 242:423426, 1988). In addition, such techniques may beutilized to change a "murine" antibody to a "human" antibody, withoutaltering the binding specificity of the antibody.

Once suitable antibodies or binding partners have been obtained, theymay be isolated or purified by many techniques well known to those ofordinary skill in the art (see Antibodies: A -Laboratory Manual, Harlowand Lane (eds.), Cold Spring Harbor Laboratory Press, 1988). Suitabletechniques including peptide or protein affinity columns, HPLC orRP-HPLC, purification on protein A or protein G columns, or anycombination of these techniques.

Another method of generating high-affinity antihippuricase antibodies isthe recombinant phage antibody system developed by Clackson et al,Nature 352:624-628 (1991), which depends upon the ability of M13 phageto exhibit functional antibody fragments as fusion proteins on theirsurface. Reagents and the phage vector pCANTAB 5 are commerciallyavailable from Pharmacia. The antibodyencoding gene is a recombinantgene consisting of the variable heavy and light encoding sequenceseparated by a linker sequence. The recombinant gene, SCFV (single-chainfragment variable) is cloned into a phagemid and expressed at the tip ofthe phage gene 3 protein in the presence of helper phage K07. Theantigen-reactive recombinant phage can be detected with anti-M13antibody conjugated to horseradish peroxidase. Recombinant phages whichproduce SCFV polypeptides with high affinity for the C. jejunihippuricase can be enriched by passage over hippuricase affinity columnsor by panning the recombinant phage library against the purified C.jejuni hippuricase. Pooled or specific clones of recombinant phageswhich produce SCFV polypeptides which react specifically with C. jejunihippuricase may be used in developing the immunoprobe. A specificrecombinant phage is anglogous to specific monoclonal antibody.

Polyclonal antibodies may be readily generated by one of ordinary skillin the art from a variety of warm-blooded animals such as horses, cows,various fowl, rabbits, mice, or rats. Briefly, a polypeptide of theinvention is utilized to immunize the animal through intraperitoneal,intramuscular, intraocular, or subcutaneous injections. An adjuvant suchas Freund's complete or incomplete adjuvant may also be employed.Following several booster immunizations, samples of serum are collectedand tested for reactivity to hippuricase. Particularly preferredpolyclonal antisera will give a signal on one of these assays that is atleast three times greater than background. Once the titer of the animalhas reached a plateau in terms of its reactivity to hippuricase, largerquantities of antisera may be readily obtained either by weeklybleedings, or by exsanguinating the animal.

High titre antisera against hippuricase may be developed in rabbits byimmunization with the purified hippuricase polypeptide of the inventionor with different hydrophilic domains of the C. jejuni hippuricase.Hippuricase-specific antibody may be purified using DEAE cellulose andaffinity columns (See Juranka, P. and VL 15 Chan, J. Biol. Chem. 260(12):7738-7743, 1985). The specificity of the antibodies can be testedby reacting with hippuricase from C. jejuni and other microorganismsthat have been shown to possess hippuricase activity.

In a preferred embodiment, a high-titre anti-enzyme serum is produced byinjecting the purified polypeptide in Tris/saline buffer and Freund'scomplete adjuvant (initial injection only) subcutaneously into the backof New Zealand White Rabbits, and once a week for 2 additional weekswith Freund's incomplete adjuvant. Booster injections with Freund'sincomplete adjuvant are given 4 weeks later and the rabbits are bled 1week after the booster through a small incision of the major veinlocated in the ears. The procedure is repeated several times with eachrabbit.

The polyclonal, monoclonal or chimeric monoclonal antibodies may be usedto detect hippuricase, parts thereof or closely related isoforms invarious samples, for example they may be used in an ELISA,radioimmunoassay or histochemical tests. Thus, the antibodies may beused to quantify the amount of hippuricase, parts thereof or closelyrelated isoforms in a sample in order to diagnose C. jejuni infections.Using methods described hereinbefore, polyclonal or monoclonalantibodies may be raised to nonconserved regions of hippuricase and usedto distinguish hippuricase from closely related isoforms and otherproteins that share a common conserved epitope.

The polyclonal or monoclonal antibodies may be labelled with adetectable marker including various enzymes, fluorescent materials,luminescent materials and 35 radioactive materials. Examples of suitableenzymes include horseradish peroxidase, biotin, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; and examples of suitable radioactive material include S-35,Cu-64, Ga-67, Zr-89, Ru-97, Tc-99m, Rh-105, Pd-109, In-111l, I-123,I-125, I131, Re-186, Au-198, Au-199, Pb-203, At-211, Pb-212 and Bi-212.The antibodies may also be labelled or conjugated to one partner of aligand binding pair. Representative examples include avidin-biotin andriboflavin-riboflavin binding protein.

Methods for conjugating or labelling the antibodies discussed above withthe representative labels set forth above may be readily accomplishedusing conventional techniques such as described in U.S. Pat. No.4,744,981 (Trichothecene Antibody); U.S. Pat. No. 5,106,951 (AntibodyConjugate); U.S. Pat. No. 4,018,884 (Fluorengenic Materials andLabelling Techniques); U.S. 20 Pat. No. 4,897,255 (Metal RadionucleotideLabeled Proteins for Diagnosis and Therapy); U.S. Pat. No. 4,988,496(Metal Radionuclide Chelating Compounds for Improved ChelationKinetics); Inman, Methods in Enzymology, Vol. 34, Affinity Techniques,Enzyme Purification; Part B, Jacoby and Wichek (eds) Academic Press, NewYork, p. 30, 1974; and Wilchek and Bayer, The Avidin-Biotin Complex inBioanalytical Applications Anal. Biochem. 171:1-32, 1988.

Any sample suspected of containing C. jejuni may be tested for thepresence or absence of hippuricase in accordance with the methods setforth herein. Samples which may be tested include bodily materials suchas blood, serum, urine, tears, saliva, feces, tissues and the like. Bothmedical and veterinary applications are contemplated. In addition tohuman samples, samples may be taken from poultry or mammals such asnon-human primates, horses, swine etc. Further, water and food samplesand other environmental samples and industrial wastes may be tested.

Before testing a sample in accordance with the methods described herein,the sample may be concentrated using techniques known in the art, suchas centrifugation and filtration. For example, a water sample of from 1to 100 ml may be subjected to centrifugation at 10,000×g for about 15minutes and the cells can be transferred to an Eppendorf tube andresedimented at 10,000×g for 5 minutes. Alternatively, the cells in thesample may be concentrated by filtration with a 0.4 μm pore size teflonfilter and resuspending in water by vortexing. The cells may be lysedwith equal volumes of 2×SDS gel-loading buffer (Sambrook et al, 1989,supra) and boiled for 5 minutes. For the hybridization and/or PCR-basedmethods described herein, nucleic acids may be extracted from cellextracts of the test sample using techniques known in the art.

In a method of the invention a predetermined amount of a sample orconcentrated sample is mixed with antibody or labelled antibody. Theamount of antibody used in the process is dependent upon the labellingagent chosen. The resulting hippuricase bound to antibody or labelledantibody may be isolated by conventional isolation techniques, forexample, salting out, chromatography, electrophoresis, gel filtration,fractionation, absorption, polyacrylamide gel electrophoresis,agglutination, or combinations thereof.

The sample or antibody may be insolubilized, for example, the sample orantibody can be reacted using known methods with a suitable carrier.Examples of suitable carriers are Sepharose or agarose beads. When aninsolubilized sample or antibody is used the hippuricase bound toantibody or unreacted antibody is isolated by washing. For example, whenthe sample is blotted onto a nitrocellulose membrane, the antibody boundto hippuricase is separated from the unreacted antibody by washing witha buffer, for example, phosphate buffered saline (PBS) with bovine serumalbumin (BSA).

When labelled antibody is used, the presence of C. jejuni can bedetermined by measuring the amount of labelled antibody bound tohippuricase in the sample or of the unreacted labelled antibody. Theappropriate method of measuring the labelled material is dependent uponthe labelling agent. For example, if the labelling agent is an enzyme,the presence of C. jejuni can be determined by measuring the enzymaticactivity using a proper enzyme substrate for colorimetric, luminescentor fluorescent systems. If the labelling agent is a fluorescentmaterial, the presence of C. jejuni can be determined by measuringfluorescence intensity, and if the labelling agent is a radioactivematerial, the presence of C. jejuni can be determined by measuring theradioactivity.

When unlabelled antibody is used in the method of the invention, thepresence of C. jejuni can be determined by measuring the amount ofantibody bound to C. jejuni using substances that interact specificallywith the antibody to cause agglutination or precipitation. Inparticular, labelled antibody against hippuricase specific antibody, canbe added to the reaction mixture. The presence of C. jejuni can bedetermined by a suitable method from among the already describedtechniques depending on the type of labelling agent. The antibodyagainst hippuricase specific antibody can be prepared and labelled byconventional procedures known in the art which have been describedherein. The antibody against hippuricase specific antibody may be aspecies specific anti-immunoglobulin antibody or monoclonal antibody,for example, goat anti-rabbit antibody may be used to detect rabbitanti-hippuricase antibody.

In an embodiment of the invention the antibody of the invention is usedas the basis of an enzyme-linked immunosorbent assay for the detectionof hippuricase. The immunoassay involves coating a solid support withpolyvalent rabbit anti-hippuricase antibody, which would capturehippuricase protein present in samples containing C. jejuni. Thecaptured hippuricase antigen can be detected using a biotinylatedaffinity purified hippuricase specific antibody. Thehippuricase-antibody-biotin complex can be readily detectedcolorimetrically using a streptavidin-alkaline phosphatase colourdevelopment system. The immunoassay may be in the form of a dip-stick.

A polypeptide of the invention may also be used to assay for a substancewhich specifically affects Campylobacter jejuni. Accordingly, theinvention provides a method for assaying for a substance that affectshippuricase activity comprising providing a polypeptide of theinvention, incubating with a substrate of the polypeptide, and a testsubstance which is suspected of affecting hippuricase, and determiningthe effect of the substance by comparing to a control. The method may beused, for example, to assay for a substance which affects the growth orthe pathogenesis of Campylobacter jejuni. Representative substrateswhich may be used in the assay are hippurate, N-Benzoyl-DL-alanine,Nα-Benzoyl-L-arginine, N-benzoyl-L-glutamic acid, N-Benzoyl-L-glycine,Nα-Benzoyl-L-histidine, N-Benzoyl-DL-leucine, N-Benzoyl-DL-methionine,N-Benzoyl-DL-phenylalanine, N-Benzoyl-L-threonine andN-Benzoyl-DL-valine.

The reagents suitable for applying the methods of the invention may bepackaged into convenient kits providing the necessary materials,packaged into suitable containers. Such kits may include all thereagents required to detect C. jejuni in a sample by means of themethods described herein, and optionally suitable supports useful inperforming the methods of the invention. In one embodiment of theinvention the kit contains a nucleotide probe which hybridizes with anucleic acid molecule of the invention, reagents required forhybridization of the nucleotide probe with the nucleic acid molecule,and directions for its use. In another embodiment of the invention thekit includes antibodies of the invention and reagents required forbinding of the antibody to C. jejuni hippuricase in a sample. In stillanother embodiment of the invention, the kit includes primers which arecapable of amplifying a nucleic acid molecule of the invention or apredetermined oligonucleotide fragment thereof, all the reagentsrequired to produce the amplified nucleic acid molecule or predeterminedfragment thereof in the polymerase chain reaction, and means forassaying the amplified sequences. The methods and kits of the presentinvention have many practical applications. For example, the methods andkits of the present invention may be used to detect C. jejuni in anymedical or veterinary sample including bodily materials such as blood,serum, urine, tears, saliva, feces, tissues and the like, samples frompoultry or mammals such as non-human primates, horses, swine etc, andwater and food samples, other environmental samples and industrialwastes.

The invention will be more fully understood by reference to thefollowing examples. However, the examples are merely intended toillustrate embodiments of the invention and are not to be construed tolimit the scope of the invention.

EXAMPLES

Materials and methods used in the examples described herein include thefollowing:

Bacterial strains and plasmids. Campylobacter jejuni ATCC43431 (strainTGH9011, serotype reference strain for 0:3) was obtained from J. L.Penner (University of Toronto, Toronto, Canada). C. jejuniATCC33560^(T), and C. coli ATCC33559^(T), C. lari and C. upsaliensiswere purchased from the American Type Culture Collection. Escherichiacoli JM101 [▴-(lac-pro) thi rspL supE endA sbcB hsdR F' traD36 proABlacI^(g) Z▴M15] (Maniatis T., Ibid.) and DH5αF'[F'endAl hsdR17(r-_(k)m+_(k)) supE44 thi-1 recAl gyrA relAl θ801acZ-N15 ▴(lacZYA-argF)₁₆₉ ]were obtained from B. McNeil (University of Toronto), E. coli DR1984 wasobtained as described in Sancar, A. et al, J. Bacteriol. 137 (1979)692-693), and Streptococcus faecalis var. zymogenes was obtained from T.Bleier (University of Toronto). Vectors used were pBR322, andpBlueScript KS-.

Media and growth conditions. C. jejuni and C. coli were grown routinelyon Brucella agar base (Difco) supplemented with 5% horse blood in a 5%CO2 incubator at 37° C. E. coli and Streptococcus faecalis were grown inLuria broth at 37° C.

Chemicals. Ampicillin, N-Benzoyl-DL-alanine, Nα-Benzoyl-L-arginine,N-benzoyl-L-glutamic acid, N-Benzoyl-L-glycine, Nα-Benzoyl-L-histidine,N-Benzoyl-DL15 leucine, N-Benzoyl-DL-methionine,N-Benzoyl-DL-phenylalanine, N-Benzoyl-L-threonine, N-Benzoyl-DL-valinewere purchased from Sigma Chemical Co. Ninhydrin was purchased fromCalbiochem, San Diego, USA. Ferric chloride was purchased from FisherScientific Corp., Fair Lawn, N.J., USA. ³² p-dATP, ³⁵ S-dATP, and ³⁵S-methionine was purchased from ICN. Restriction enzymes were purchasedfrom Boehringer Mannheim, Germany, Gibco-BRL, Mississauga, Canada, andPharmacia, Upsala, Sweden. Sequencing was performed with the Sequenasekit from United States Biochem. Co., Ohio, USA. Nick-translation wasperformed using the nick-translation kit from BRL, Mississauga, Canada.

Preparation of crude extracts. C. jejuni ATCC43431, C. coli ATCC33559,Streptococcus faecalis var. zymogenes, E. coli JM101, and E. coli JM101containing recombinant plasmids were grown overnight in their respectivemedia supplemented with 1% hippuric acid and 50 μg/ml ampicillin whereappropriate. Cells were harvested at 12,000×g for 30 min., washed twicein 20 ml 0.85% NaCl, and resuspended in 2 ml 0.15 M potassium phosphatebuffer pH 7.4. The cell suspension was passed through a French presstwice at a pressure of 16,000 lbs./sq.in. The extract was cleared ofcell envelope by centrifugation 20,000×g for 40 min., and stored frozenat -20° C.

DNA manipulation. Preparation of plasmid DNA from E. coli was by rapidalkaline lysis procedure, and digestion with restriction endonucleases,ligation with T4 DNA ligase, transformation with recombinant plasmids,and agarose gel electrophoresis were done as described in Sambrook etal, 1989, supra. Genomic C. jejuni DNA was prepared, digested, andblotted as described in Chan et al, Gene 73:185-191, 1988.

Probe preparation and blot hybridization. Plasmid DNA was labelled usinga nick-translation kit from Bethesda Research Laboratories (Burlington,Ontario, Canada) using [α-³² P]dATP. Plasmid restriction fragments wereisolated using the GeneCleanII kit. Nick-translated DNA was isolatedfrom unincorporated nucleotides through a Sephadex-G50 spun column. Thespecific activity of the probes was approximately 1.7×10⁷ dpm/μg DNA.Hybridization was performed as described in Sambrook et al, 1989, supra.

Nucleotide sequencing. The DNA sequence was determined by the dideoxychain termination method described by Sanger et al. (Sanger et al. PNASUSA, 74:5463, 1977). Small-scale plasmid preparations were obtained bythe alkaline lysis procedure (Maniatis, Ibid.) from cells grownovernight in 1.5 ml cultures, and sequenced using the Sequenase kit.Nested deletion clones for sequencing were generated by the method ofHenikoff, Gene 28: 351-359, 1984, which is incorporated herein byreference.

Example 1

Isolation, Mapping and Sequencing of Hippuricate Hydrolyzing Clone

A C. jejuni genomic library was constructed in pBR322 following themethods of Chan et al (Gene 73: 185-191, 1988). More particularly, C.jejuni DNA was isolated from strain TGH9011 (serotype 0:3) for libraryconstruction. C. jejuni stock cultures were maintained at -70° C. inglycerol salt solutions (40%, v/v, glycerol-3%, w/v, Na3. citrate).Cells were grown on 5% horse blood agar plates with Columbia agar base(Oxoid Ltd., London, U.K.) and incubated for 48 hours at 37° C. in 5%CO2.

C. jejuni genomic DNA was isolated and partially digested generallyfollowing the procedure described in Maniatis et al, Molecular Cloning,A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1982). Partial Sau3A digest conditions which gave the greatestmass of C. jejuni DNA fragments in the 4-9 kb range were determined, andlarge-scale preparation of partially digested DNA (250 μg) was carriedout. The reaction was stopped by the addition of EDTA to 20 mM and thetube was placed in ice. After two extractions with phenol-chloroform andethanol precipitation, the DNA was redissolved in 500 μl TE, loaded on a10-40% sucrose gradient and centrifuged in a Beckman rotor SW27 at 26000rev./min for 20 hours at 20° C. Fractions of 0.5 ml were collected andDNA sizes checked on an agarose gel. The 20 appropriate fractions werepooled and dialysed against 4 liters of TE (10 mM Tris-HCl pH 8, 1 mMEDTA). After butanol extraction and ethanol precipitation the DNA wasredissolved in TE at 0.5 μg/ml.

Plasmid pBR322 (1 μg) was completely digested with 5 units of BamHI at37° C. for 200 minutes before the end of the restriction enzyme digest,approximately 6-8 units of calf intestine alkaline phosphatase(Boehringer Mannheim) were added to the digest which was incubated foranother 20 minutes at 37° C. The reaction was stopped by heating at 65°C. in the presence of 20 mM EDTA and 0.5% SDS. The DNA was thenphenol-extracted, ethanol-precipitated and redissolved in TE to aconcentration of 1 μg/μl.

BamHI-cut and dephosphorylated pBR322 (1 μg) was ligated to 0.5 μg ofthe C. jejuni DNA fragments using 1 unit (Weiss) of T4 DNA ligase in theappropriate buffer for 16 hours at 14° C. E. coli MM294 was transformedwith 100 ng of the ligation mixture by the established proceduredetailed in Hanahan, J. Mol. Biol. 166 (1983) 557-580.

The resulting library of 6.3×104 recombinant plasmids was amplified byinoculating the whole culture into 30 ml LB broth containing 50 μgAp/ml, and after a 10-hour incubation at 37° C. the culture which thenhad a titer of 2×109 cells/ml was frozen in 50% glycerol in 1 mlaliquots. The number of tetracycline-resistant colonies in this culturewas less than 0.2%.

Recombinant plasmids yielding hippurate hydrolysis activity in E. coliwere isolated from the C. jejuni genomic library, prepared as describedin Example 1. Clones were screened for hippuricase activity by theirability to catalyze the conversion of N-benzoylglycine to benzoic acidand glycine, the hallmark of hippuricase activity. One thousand fivehundred clones were screened in groups of ten to twenty and rescreenedusing individual colonies. One clone designated pHIPPO containing theputative hippuricase gene was isolated as positive for glycineformation. Further analysis of this clone demonstrated the production ofthe second product benzoic acid.

The recombinant plasmid pHIPPO was examined by restriction enzymeanalysis, and analysis of deletion subclones to determine the locationof the region encoding hippuricase activity. DNA was prepared from therecombinant plasmids and analyzed by restriction endonuclease digestionusing the restriction enzymes BamHI (B), BglII (G), ClaI (C), EcoRI (E),EcoRV (V), NindIII (H), PstI (P), RsaI (R), SspI (S), XbaI (X), andXhoII (O). There were no restriction enzyme sites for BamHI, EcoRI,EcoRV, or PstI in the insert. The restriction map of recombinant plasmidpHIPPO is shown in FIG. 2.

A 4.0 kb ClaI fragment from pHIPPO was subcloned in both orientations inthe ClaI site in pBlueScript, which retained hippuricase activity.Further subclones defined the region encoding hippuricase activity to a2.7 kb region in the 5.0 kb C. jejuni DNA insert. The region wassequenced on both strands. The DNA sequence was determined by thedideoxy chain termination method 5 described by Sanger et al. (Sanger,Supra). Small-scale plasmid preparations were obtained by the alkalinelysis procedure (Maniatis, Supra) from cells grown overnight in 1.5 mlcultures, and sequenced using the Sequenase kit (United States BiochemCo., Ohio, U.S.A.). The region encoding hippuricase activity wassequenced on both strands using the Sequenase kit. The Sequence is shownin SEQ I.D. NO:1 and NO:2 and FIG. 1. The hippuricase gene shows nosignificant homology to any gene in the GenBank database, nor does thetranslated polypeptide show similarity to any other protein sequence.The gene is 1.338 kb in size and generates a theoretical product of 446amino acids with a deduced molecular weight of 50 kd. the ccdon usage ofthe hippuricase gene is consistent with that of other Campylobacterjejuni chromosomal genes 20 sequenced by the inventors.

Deletion mutants for sequencing were constructed using SacI and XbaI inone direction as described by Henikoff, Gene 28: 351-359, 1984.

Southern hybridizations were performed to determine the location of therelevant gene on the specific restriction enzyme fragments used togenerate the physical map of C. jejuni TGH9011. These include SalI,SmaI, and SacII restriction fragments. C. jejuni genomic DNA wasdigested with the restriction enzymes, and transferred by Vacuum Blotonto GeneScreen nylon membrane (Kim et al, J. Bacteriol 174:3494-3498).A SmaI-HindIII fragment of pHIPPO, containing only hippuricase encodingsequence was labelled with ³² p by nick translation as described aboveand used to probe the genomic filter containing pulsed-field gelelectrophoresis separated fragments of C. jejuni genomic DNA. Asillustrated in FIGS. 2 and 3, there is only one copy of the gene in C.jejuni, and it is located in a region of the chromosome bounded by theSalI A (1,050 kb), SacII A (420 kb) and SmaI A (465 kb) fragments of thephysical map shown in FIG. 3. The gene is therefore located within a 420kb region of the chromosome as are the arg H and rrl genes.

Example 2

Analysis of the protein expressed by the hippuricase gene

The protein product of the hippuricase gene was analyzed by maxicellanalysis as follows. Plasmid encoded proteins were labelled inW-irradiated E. coli DR1984 cells generally as described by Sancar et al(J. Bacteriol. 137:692-693, 1979 and J. Mol. Biol. 148:45-62, 1981).Cells were W-irradiated with a germicidal lamp 15 (15 W) at a height of50 cm. Survival was between 10-4 and 10-5 following 12-15 h incubationwith 200 μg/ml Dcycloserine. Irradiated cells were washed two times withHershey salts and then labelled with 35S-methionine (40 μCi/ml) for 1 hrin Hershey medium. Cells were lysed by boiling for 3 min. in 50 μl 2×LSBand labelled proteins were separated by 0.1% SDS-13% PAGE as describedby Laemmli (1970). After electrophoresis, gels were stained withCoomassie brilliant blue R-250, dried onto 3 MM cellulose paper and thenexposed overnight to Kodak XAR-5 film at -70° C. Controls includedDR1984 with no plasmid, and DR1984 with pBlueScript. The DR1984 strainshowed no proteins produced, the pBlueScript plasmid produced a 30 kDampicillin resistance determinant. The recombinant plasmid produced theampicillin resistance determinant, and an additional 42 kD productcorresponding to the hippuricase gene product. FIG. 4 shows the resultsof the maxicell analysis and confirms that the clone is expressed in E.coli.

Example 3

Species Specificity of the Hippuricase Protein

The ability of a hippuricase probe, to detect other species ofCampylobacter was examined.

An 800 bp SphI/HindIII fragment of plasmid pHIPPO-C/Spl.7 containing thehippuricase encoding sequence (See FIG. 2) was used as ahippuricase-specific probe. Genomic DNAs of hippuricase positive(type-strain) and hippuricase negative strains (5) of C. jejuni weredigested with HindIII, separated by gel electrophoresis, transferred toGeneScreen, and probed with a radiolabelled probe as described byManiatis et al (1982, supra).

The hippuricase probe specifically labelled a 2.2 kb HindIII band in thetype-strain for C. jejuni (lane 1). A single hybridizing band was alsoobserved for all the hippuricase C. jejuni isolates (lanes 2-6), rangingin size from 2.2 to 2.4 kb in size. Therefore, all the C. jejuni strainscontained the hippuricase gene sequences, even if they werebiochemically negative (FIG. 5). One of the HindIII sites is locatedwithin the hippuricase gene, but the second is located upstream of thehippuricase gene. This results in a polymorphic banding pattern usingthis restriction enzyme. Southern hybridization is being performed usingSau3A digested chromosomal DNA which should generate a common 670 bpfragment.

The same probe was used to screen various C. jejuni and C. coli isolatesusing the method described above. Some of the isolates were type strainsand some were classified using conventional methods of biochemicalreactions etc. As shown in FIG. 6, the type-strain for C. jejuni waspositive (lane 1), generating a 2.2kb HindIII hybridizing band. Thetype-strain for C. coli was negative, even under low stringencyconditions (25 C, 2×SSC) of hybridization. All the C. jejuni strainsgave a positive hybridizing signal with the hippuricase probe (lanes10-14), and were positive in the biochemical test. Seven C. coliisolates, which were not type strains, gave a weak hybridizing signalusing a C. jejuni hippuricase-specific probe at high stringency (65 C,0.2×SSC) (FIG. 6). These isolates were all negative in the hippuricasebiochemical test and were incorrectly identified as C. coli.

Example 4

Demonstration of Hippuricase in Cell-free Extracts

Crude cell-free extracts were prepared from the recombinants andhippuricase activity was measured. More particularly, C. jejuniATCC43431, C. coli ATCC33559, Streptococcus faecalis var. zymogenes, E.coli JM101, and E. coli JM101 containing recombinant plasmids were grownovernight in their respective media supplemented with 1% hippuric acidand 50 llg/ml ampicillin where appropriate. Cells were harvested at12,000×g for 30 min., washed twice in 20 ml 0.85% NaCl, and resuspendedin 2 ml 0.15 M potassium phosphate buffer pH 7.4 . The cell suspensionwas passed through a French press twice at a pressure of 16,000lbs./sq.in. The extract was cleared of cell envelope by centrifugationat 20,000×g for 40 min., and stored frozen at -20° C.

Enzyme activity in the crude cell free extracts was determined asfollows. Hydrolytic activity towards N-benzoylamino-acid was thatestimated by the procedure of Kameda et al. (Chem. Pharm. Bull. Tokyo16:1023, 1968). The reaction mixture contained 1 ml of 10 mMN-benzoylamino-acid, 0.5 ml of 0.1 M Tris buffer (pH 8.0), and 0.5 ml ofappropriately diluted enzyme solution. Incubation was at 37° C. for 0,10, and 20 min., after which 0.2 ml was removed and boiled for 2 min. toeliminate enzyme activity. A 0.1 ml aliquot of ninhydrin reagent (3.5%in a 1:1 solution of acetone/butanol) was added, and boiled for 20 min.The solution was diluted with 60% ethanol, and the absorption intensitywas measured at 570 nm relative to the amino acid standard. The enzymeactivity is expressed in terms of micromoles of liberated amino acid perhr. per mg protein under the above conditions. Protein content of theenzyme solution was measured by the method of Lowry et al., J. Biol.Chem. 193, 265-275, 1951, which is incorporated herein by reference,using bovine serum albumin as the standard.

Hippuricase activity was detected by the following methods: (1) One mlof overnight culture was centrifuged at 12000×g for 2 min. at roomtemperature, as were all subsequent centrifugations. The bacterialpellet was washed in a 1% aqueous solution of sodium hippurate pH 7.2,recentrifuged, and resuspended in 100 μl of 1% aqueous sodium hippurate.The cells were incubated at 37 C for 1 hr. and pelleted. The supernatantwas transferred to a 96 well microtitre plate and assayed for thepresence of glycine by the addition of 50 μl ninhydrin reagent (3.5% in1:1 acetone/butanol) according to the method of Hwang and Ederer (J.Clin. Microbiol. 1:114, 1975). (2) Benzoic acid was detected using theprocedure described by Facklam et al. (Appl. Microbiol. 27:107, 1974).After overnight growth in Luria broth supplemented with 1% hippurate,the cells were centrifuged, and 0.8 ml of the clarified supernatant wasmixed with 0.2 ml acidified FeC13, and the presence of a large brownprecipitate was considered positive; (3) Alkaline reaction was detectedin phenol-red hippurate agar slants as proposed by Thirst (J. Gen.Microbiol. 17:390, 1957). The media contains casamino acids, 1.0 g;yeast extract, 1.0 g; K₂ HPO₄, 10 mM; NaCl, 5.0 g; sodium hippurate, 10g; phenol red (1.6% solution), 2 ml; agar, 13 g; distilled water, 1liter. Positive controls in each assay were Streptococcus faecalis var.zymogenes, and Campylobacter jejuni. Negative controls were uninoculatedbroth cultures, E. coli JM101, E. coli MM294, and Campylobacter coli.

The enzyme was detectable in C. jejuni and Streptococcus faecalis var.zymogenes, but not in E. coli or C. coli. Hippuricase activity waslinear with respect to protein concentration and time, indicating thatthe protein functions as an enzyme. The results also confirm thathippuricase activity is not associated with the cell wall or cellmembrane fraction of the bacterial organism. The C. jejuni protein wasnot denatured by incubation at 65° C., but was inactivated at 100° C.after a two minute 35 exposure.

The results show that the genetic locus encoding hippuricase is specificfor C. jejuni, but is not detectable by enzymatic or DNA hybridization(see Example 3) in any other species of Campylobacter. Hippuricaseactivity is present in S. faecalis var. zymogenes, however, the probe(See Example 3) is unable to detect 5 hippuricase sequences form thisorganism

Example 5

Substrate specificity of the recombinant hippuricase protein

Substrate specificity of the recombinant hippuricase protein wascompared to that of the protein from the cell extract of C. jejuni. Therecombinant hippuricase showed greatest activity in benzoyl(bz-)glycineand bz-histidine, whereas the cell extract C. jejuni hippuricase hadgreatest activity with bz-leucine and bz-methionine. The recombinantprotein did not show any activity with bz-arginine, in contrast to theweak reaction with the cell extract from C. jejuni.

Example 6

The glutathione S-transferase gene fusion system is an integrated systemfor the expression, purification, and detection of fusion proteinsproduced in E. coli. The vectors offer a tac promoter for chemicallyinducible, high-level expression; an internal lac Iq gene for use in anyE. coli host; mild elution conditions for release of fusion proteinsfrom the affinity matrix, thus minimizing effects on antigenicity andfunctional activity; and thrombin cleavage recognition sites forcleaving the desired protein from the fusion product.

A fusion of the hippuricase gene was constructed into this vector byligation of an XmnI restriction enzyme fragment containing thecarboxy-terminal portion of the gene into the SmaI restriction site inthe vector. The reading frame of hippuricase is maintained with respectto GST. Forty-seven nucleotides upstream of the XmnI restriction enzymesite and without the coding sequence from hippuricase were deleted inthis construct. This represents a loss of only sixteen amino-terminalamino-acid residues from the native polypeptides, which should notinterfere with the production of antibodies to the hippuricase protein.The construct has the following characteristics at the fusion junction:

    Thombin cleavage site                                                         ... CTG GTT CCG CGT                                                            ... Leu Val Pro Arg                                                           GST                                                                            -        GGA TCC CAA TTC GTC ATC AAA TTC...                                             Gly Ser Gln Phe Val Ile Lys Phe...                                  Hippuricase                                                             

These sequences were confirmed by dideoxynucleotide sequencing of thejunction region using a synthetic oligonucleotide primer designedspecifically for this purpose.

The production of a fusion protein of the expected size was demonstratedfor a GST-hippuricase protein in whole cell extracts of E. coli inducedwith IPTG. The GST-hippuricase protein was purified to homogeneity byusing a Glutathione Sepharose affinity matrix to separate the fusionprotein from total cellular protein, and by elution of the fusionprotein using reduced glutathione.

The hippuricase protein can be removed from the GST affinity handle bycleavage of the fusion protein with thrombin, followed by removal of theGST moiety using the Glutathione Sepharose system used to initiallyisolate the fusion protein from total protein.

Legend for FIG. 3

Location of the hippuricase gene on the physical map of C. jejuni strainTGH9011 with respect to other genetic markers isolated from thisorganism. The 1812 kb genome is divided into quartiles using rrnA as thezero-reference point. Genes which have been mapped onto the chromosomearc listed below.

    ______________________________________                                        Gene symbol   Phenotypic trait                                                ______________________________________                                        hipO          Hippuricase                                                       rrlA 23S rRNA of rrnA operon                                                  rrsA 16S rRNA of rrnA operon                                                  rrlB 23S rRNA of rrnB operon                                                  rrsB 16S rRNA of rrnB operon                                                  rrlC 23S rRNA of rrnC operon                                                  rrsC 16S rRNA of rrnC operon                                                  argA NAc-glutamate synthetase                                                 argB NAc-glutamate kinase                                                     argC NAc-glutamylphosphate reductase                                          argD Acetylornithine aminotransferase                                         argE Acetylornithase                                                          argF Ornithine caramoyltransferase                                            argH Argininosuccinate lyase                                                  fla1 fla2 Flagellin                                                           glyA Serine hydroxymethyltransferase                                          lysS Lysyl tRNA sythetase                                                     proA Glutamylphosphate reductase                                              flgG Flagellin-associated protein                                           ______________________________________                                    

Legend for FIG. 4

Maxicell analysis of hippuricase-encoding plasmids.

Plasmid encoded proteins were labelled in UV-irradiated E. coli DR1984cells as described by Sancar et al. (1979, 1981). Cells wereUV-irradiated with a germicidal lamp (15 W) at a height of 50 cm.Survival was between 10⁻⁴ and 10⁻⁴ following 12-15 h incubation with 200μg/ml D-cycloserine. Irradiated cells were washed two times with Hersheysalts and then labelled with 35S-methionine (40 μCi/ml) for 1 h inHershey medium. Cells were lysed by boiling for 3 min in 50 μl 2×LSB andlabelled proteins were separated by 0.1% SDS-13% PAGE as described byLaemmli (1970). After electrophorcsis, gels were stained with Coomassicbrilliant blue R-250, dried onto 3 MM cellulose paper and then exposedto Kodak XAR-5 film at -70° C. E. coli control cells containing noplasmid produced no radioactively labelled proteins. Cells containingplasmid pBR322 produced a single protein of molecular weight 31 kd,corresponding to the ampicillin resistance determinant. Cells containingplasmid pHIPO produced both a 31 kd protein and a 42 kd proteincorresponding to the hippuricase polypeptide product.

Legend for FIG. 5

Southern hybrdization of chromosomal DNA of hippuricase-negativeisolates of Campylobacter jejuni.

Genomic DNA was prepared from five strains of C. jejuni which were shownto be hippuricase negative in the standard biochemical test, and thetype-strain for C. jejuni which is hippuricase positive. The DNA wasdigested with HindIII. The fragments were separated by gelelectrophoresis, transferred to GeneScreen, and probed with aradiolabelled 800 bp kb SphI/HindIII fragment of pHIP-C/Spl.7 asdescribed by Maniatis et al. (1982).

The hippuricase probe specifically labelled a 2.2 kb HindIII band in thetype strain for C. jejuni (lane 1). A single hybridizing band was alsoobserved for all the hippuricase negative C. jejuni isolates (lanes2-6), ranging in size from 2.2 to 2.4 kb in size. The polymorphic natureof the hybridizing pattern is a result of the fact that one of theHindIII sites is not within the encoding sequence of the hippuricasegene.

Legend for FIG. 6

Southern hybrdization of chromosomal DNA of Campylobacter coli andCampylobacter jejuni isolates.

Genomic DNA was prepared from the type-strains of C. jejuni, and C.coli, and from strains corresponding to serotype-reference strains. TheDNA was digested with HindIII. The fragments were separated by gelelectrophoresis, transferred to GeneScreen, and probed with aradiolabelled 800 bp kb SphI/HindIII fragment of pHIP-C/Spl.7 asdescribed by Maniatis et al. (1982).

The hippuricase probe specifically labelled a 2.2 kb HindIII band in thetype strain for C. jejuni (lane 1), but did not detect any hybridizingband in C. coli, even at low stringency (25 C, 2×SSC). All C. jejunistrains hybridized to the hippuricase probe (lanes 10-14). All C. coliserotype reference strains also hybridized to the hippuricase probe athigh stringency (65 C, 0.2×SSC), although they generated a weakersignal.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 2                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1338 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1338                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - ATG AGT GAC TTT TGG AGA ATA CCT TTG TTA TA - #C TAT CAT TTA ACC        ATG       48                                                                    Met Ser Asp Phe Trp Arg Ile Pro Leu Leu Ty - #r Tyr His Leu Thr Met            1               5 - #                 10 - #                 15              - - GGT AGT AAA TCT TGG AGT GTT GAT AAA ACC CA - #T AGA TTT ACT TTG GGT           96                                                                       Gly Ser Lys Ser Trp Ser Val Asp Lys Thr Hi - #s Arg Phe Thr Leu Gly                        20     - #             25     - #             30                  - - TTT GTT TAT ATT TTT GCT TTG ATT TTT ATT TC - #A GCG ATC TTA GCA CAA          144                                                                       Phe Val Tyr Ile Phe Ala Leu Ile Phe Ile Se - #r Ala Ile Leu Ala Gln                    35         - #         40         - #         45                      - - TTT GTT TTA CCT AGA AGA GAA AAT TTA TAC AA - #G GAG AAA AAT AGA TTG          192                                                                       Phe Val Leu Pro Arg Arg Glu Asn Leu Tyr Ly - #s Glu Lys Asn Arg Leu                50             - #     55             - #     60                          - - AAT TTA ATT CCA GAA ATA CTA GAC TTA CAA GG - #C GAA TTT GAA AAA ATT          240                                                                       Asn Leu Ile Pro Glu Ile Leu Asp Leu Gln Gl - #y Glu Phe Glu Lys Ile            65                 - # 70                 - # 75                 - # 80       - - CGT CAT CAA ATT CAT GAA AAT CCT GAG CTT GG - #T TTT GAT GAA TTA TGT          288                                                                       Arg His Gln Ile His Glu Asn Pro Glu Leu Gl - #y Phe Asp Glu Leu Cys                            85 - #                 90 - #                 95              - - ACT GCA AAA TTA GTG GCG CAA AAA TTA AAA GA - #A TTT GGT TAT GAG GTT          336                                                                       Thr Ala Lys Leu Val Ala Gln Lys Leu Lys Gl - #u Phe Gly Tyr Glu Val                       100      - #           105      - #           110                  - - TAT GAG GAA ATA GGA AAA ACA GGC GTT GTG GG - #G GTT TTA AAA AGG GGA          384                                                                       Tyr Glu Glu Ile Gly Lys Thr Gly Val Val Gl - #y Val Leu Lys Arg Gly                   115          - #       120          - #       125                      - - ATA GCG ATT AAA AAA ATA GGA CTC GTG CAG AT - #A TGG AAT GCT TTG CCT          432                                                                       Ile Ala Ile Lys Lys Ile Gly Leu Val Gln Il - #e Trp Asn Ala Leu Pro               130              - #   135              - #   140                          - - TTG CAA GAA TGC ACA AAT TTG CCT TAT AAA AG - #C AAA AAA GAA AAT GTA          480                                                                       Leu Gln Glu Cys Thr Asn Leu Pro Tyr Lys Se - #r Lys Lys Glu Asn Val           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - ATG CAT GCT TGC GGT CAT GAT GGA CAT ACT AC - #T TCT TTA TTG CTT        GCT      528                                                                    Met His Ala Cys Gly His Asp Gly His Thr Th - #r Ser Leu Leu Leu Ala                          165  - #               170  - #               175              - - GCA AAG TAT TTA GCA AGT CAG AAT TTT AAT GG - #C ACT TTA AAT CTT TAT          576                                                                       Ala Lys Tyr Leu Ala Ser Gln Asn Phe Asn Gl - #y Thr Leu Asn Leu Tyr                       180      - #           185      - #           190                  - - TTT CAA CCT GCT GAA GAG GGT TTG GGT GGT GC - #T AAG GCA ATG ATA GAA          624                                                                       Phe Gln Pro Ala Glu Glu Gly Leu Gly Gly Al - #a Lys Ala Met Ile Glu                   195          - #       200          - #       205                      - - GAT GGA TTG TTT GAA AAA TTT GAT AGT GAT TA - #T GTT TTT GGA TGG CAC          672                                                                       Asp Gly Leu Phe Glu Lys Phe Asp Ser Asp Ty - #r Val Phe Gly Trp His               210              - #   215              - #   220                          - - AAT ATG CCT TTT GGT AGC GAT AAG AAA TTT TA - #T CTT AAA AAA GGT GCG          720                                                                       Asn Met Pro Phe Gly Ser Asp Lys Lys Phe Ty - #r Leu Lys Lys Gly Ala           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - ATG ATG GCT TCT TCG GAT AGT TAT AGC ATT GA - #A GTT ATT GGA AGA        GGT      768                                                                    Met Met Ala Ser Ser Asp Ser Tyr Ser Ile Gl - #u Val Ile Gly Arg Gly                          245  - #               250  - #               255              - - GGT CAT GCA AGT GCT CCA AAA AAA ATA ACA GA - #T CCT ATT TAT GCT GCT          816                                                                       Gly His Ala Ser Ala Pro Lys Lys Ile Thr As - #p Pro Ile Tyr Ala Ala                       260      - #           265      - #           270                  - - TCT TTA CTT ATT GTA ACT TTA CAA AGC ATA GT - #A TCT TGC AAT GTT GAT          864                                                                       Ser Leu Leu Ile Val Thr Leu Gln Ser Ile Va - #l Ser Cys Asn Val Asp                   275          - #       280          - #       285                      - - CCC CAA AAT TCA GCA GTT GTA AGC ATA GGA GC - #T TTT AAT GCT GGA CAT          912                                                                       Pro Gln Asn Ser Ala Val Val Ser Ile Gly Al - #a Phe Asn Ala Gly His               290              - #   295              - #   300                          - - GCT TTT AAT ATC ATT CCA GAT ATT GTA ACG AT - #T AAA ATG AGT GTT AGA          960                                                                       Ala Phe Asn Ile Ile Pro Asp Ile Val Thr Il - #e Lys Met Ser Val Arg           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - GGA TTA GAT AAT GAA ACT AGA AAG CTA ACT GA - #A GAA AAA AAA AAT        AAA     1008                                                                    Gly Leu Asp Asn Glu Thr Arg Lys Leu Thr Gl - #u Glu Lys Lys Asn Lys                          325  - #               330  - #               335              - - ATT TGT AAA GGT CTT GCA CAG GCT AAT GAT AT - #A GAG ATT AAA ATC AAT         1056                                                                       Ile Cys Lys Gly Leu Ala Gln Ala Asn Asp Il - #e Glu Ile Lys Ile Asn                       340      - #           345      - #           350                  - - AAA AAT GTT GTT GCA CCA GTG ACT ATG AAT AA - #C GAT GAA GCT GTG GAT         1104                                                                       Lys Asn Val Val Ala Pro Val Thr Met Asn As - #n Asp Glu Ala Val Asp                   355          - #       360          - #       365                      - - TTT GCT AGT GAG GTT GCA AAA GAA TTA TTT GG - #C GAA AAA AAT TGT GAA         1152                                                                       Phe Ala Ser Glu Val Ala Lys Glu Leu Phe Gl - #y Glu Lys Asn Cys Glu               370              - #   375              - #   380                          - - TTT AAT CAT CGT CCT TTA ATG GCA AGT GAG GA - #T TTT GGA TTT TTT TAC         1200                                                                       Phe Asn His Arg Pro Leu Met Ala Ser Glu As - #p Phe Gly Phe Phe Tyr           385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - GAA ATG AAA AAA TGT GCC TAT GCT TTT TTA GA - #A AAT GAA AAC GAC        ATT     1248                                                                    Glu Met Lys Lys Cys Ala Tyr Ala Phe Leu Gl - #u Asn Glu Asn Asp Ile                          405  - #               410  - #               415              - - TAT TTA CAT AAT TCT AGT TAT GTT TTT AAT GA - #T AAG CTT TTA GCT AGG         1296                                                                       Tyr Leu His Asn Ser Ser Tyr Val Phe Asn As - #p Lys Leu Leu Ala Arg                       420      - #           425      - #           430                  - - GCT GCA AGT TAT TAT GCG AAG CTA GCT TTA AA - #A TAC TTA AAA                 - #1338                                                                    Ala Ala Ser Tyr Tyr Ala Lys Leu Ala Leu Ly - #s Tyr Leu Lys                           435          - #       440          - #       445                      - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 446 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Ser Asp Phe Trp Arg Ile Pro Leu Leu Ty - #r Tyr His Leu Thr Met        1               5 - #                 10 - #                 15              - - Gly Ser Lys Ser Trp Ser Val Asp Lys Thr Hi - #s Arg Phe Thr Leu Gly                   20     - #             25     - #             30                  - - Phe Val Tyr Ile Phe Ala Leu Ile Phe Ile Se - #r Ala Ile Leu Ala Gln               35         - #         40         - #         45                      - - Phe Val Leu Pro Arg Arg Glu Asn Leu Tyr Ly - #s Glu Lys Asn Arg Leu           50             - #     55             - #     60                          - - Asn Leu Ile Pro Glu Ile Leu Asp Leu Gln Gl - #y Glu Phe Glu Lys Ile       65                 - # 70                 - # 75                 - # 80       - - Arg His Gln Ile His Glu Asn Pro Glu Leu Gl - #y Phe Asp Glu Leu Cys                       85 - #                 90 - #                 95              - - Thr Ala Lys Leu Val Ala Gln Lys Leu Lys Gl - #u Phe Gly Tyr Glu Val                  100      - #           105      - #           110                  - - Tyr Glu Glu Ile Gly Lys Thr Gly Val Val Gl - #y Val Leu Lys Arg Gly              115          - #       120          - #       125                      - - Ile Ala Ile Lys Lys Ile Gly Leu Val Gln Il - #e Trp Asn Ala Leu Pro          130              - #   135              - #   140                          - - Leu Gln Glu Cys Thr Asn Leu Pro Tyr Lys Se - #r Lys Lys Glu Asn Val      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Met His Ala Cys Gly His Asp Gly His Thr Th - #r Ser Leu Leu Leu        Ala                                                                                             165  - #               170  - #               175             - - Ala Lys Tyr Leu Ala Ser Gln Asn Phe Asn Gl - #y Thr Leu Asn Leu Tyr                  180      - #           185      - #           190                  - - Phe Gln Pro Ala Glu Glu Gly Leu Gly Gly Al - #a Lys Ala Met Ile Glu              195          - #       200          - #       205                      - - Asp Gly Leu Phe Glu Lys Phe Asp Ser Asp Ty - #r Val Phe Gly Trp His          210              - #   215              - #   220                          - - Asn Met Pro Phe Gly Ser Asp Lys Lys Phe Ty - #r Leu Lys Lys Gly Ala      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Met Met Ala Ser Ser Asp Ser Tyr Ser Ile Gl - #u Val Ile Gly Arg        Gly                                                                                             245  - #               250  - #               255             - - Gly His Ala Ser Ala Pro Lys Lys Ile Thr As - #p Pro Ile Tyr Ala Ala                  260      - #           265      - #           270                  - - Ser Leu Leu Ile Val Thr Leu Gln Ser Ile Va - #l Ser Cys Asn Val Asp              275          - #       280          - #       285                      - - Pro Gln Asn Ser Ala Val Val Ser Ile Gly Al - #a Phe Asn Ala Gly His          290              - #   295              - #   300                          - - Ala Phe Asn Ile Ile Pro Asp Ile Val Thr Il - #e Lys Met Ser Val Arg      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Gly Leu Asp Asn Glu Thr Arg Lys Leu Thr Gl - #u Glu Lys Lys Asn        Lys                                                                                             325  - #               330  - #               335             - - Ile Cys Lys Gly Leu Ala Gln Ala Asn Asp Il - #e Glu Ile Lys Ile Asn                  340      - #           345      - #           350                  - - Lys Asn Val Val Ala Pro Val Thr Met Asn As - #n Asp Glu Ala Val Asp              355          - #       360          - #       365                      - - Phe Ala Ser Glu Val Ala Lys Glu Leu Phe Gl - #y Glu Lys Asn Cys Glu          370              - #   375              - #   380                          - - Phe Asn His Arg Pro Leu Met Ala Ser Glu As - #p Phe Gly Phe Phe Tyr      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Glu Met Lys Lys Cys Ala Tyr Ala Phe Leu Gl - #u Asn Glu Asn Asp        Ile                                                                                             405  - #               410  - #               415             - - Tyr Leu His Asn Ser Ser Tyr Val Phe Asn As - #p Lys Leu Leu Ala Arg                  420      - #           425      - #           430                  - - Ala Ala Ser Tyr Tyr Ala Lys Leu Ala Leu Ly - #s Tyr Leu Lys                      435          - #       440          - #       445                    __________________________________________________________________________

We claim:
 1. A purified and isolated polypeptide having an amino acidsequence as shown in SEQ ID NO:2.
 2. A purified and isolated polypeptideencoded by a nucleic acid sequence as shown in SEQ ID NO:1 wherein Talso can be U.
 3. A purified and isolated polypeptide that has enzymaticactivity of Campylobacter jejuni hippuricase and an amino acid sequenceas shown in SEQ ID NO:2.